Aptamer is a single-stranded RNA or DNA sequence that binds to target materials (e.g. small molecules, proteins, intact viruses, or cells) with high specificity and affinity. For biosensor applications, antibodies have been often used for detecting molecules due to their good specific binding property. Compared with antibodies, aptamers have several outstanding features; good stability, simple synthesis, high sensitivity, wide applicability, high resistance against denaturation, and easy labeling. Because of these properties, aptamers have received much attention as excellent substitutes for antibodies and they have been employed in various sensing methods of biosensors, including surface plasmon resonance, optical detection, quartz crystal microbalance, calorimetric detection and electrochemistry. Among those forementioned sensing methods, the electrochemical method has the properties of high sensitivity, inherent simplicity, portability, and low cost. As for improving the electrode material characteristics, Zinc oxide (ZnO) nanostructures have been considered because of many advantages such as nontoxicity, biological compatibility, fast electron transfer rate, and easy preparation. Especially, the high isoelectric point (IEP, ~9.5) of nanostructured ZnO makes it suitable for immobilizing proteins with low IEP. At the physiological pH value of 7.4, the positively charged ZnO nanorods matrix not only binds well with negatively charged proteins (e.g. thrombin), but also promotes electron transfer between ferrocene (Fc) attached to the aptamer and the Au electrode, when the ferrocene undergoes the electrochemical reaction with the target material. Therefore, an electrochemical sensor, utilizing ZnO nanorods electrode and an aptamer, has been realized and its characteristics have been investigated in this study.
For this immunosensor, thrombin was selected as a target material. Thrombin exists in blood and tissue and it is a specific serine protease involved in the coagulation cascade, which converts soluble fibrinogen into insoluble strands of fibrin and catalyzes many other coagulation-related reactions. The concentration of thrombin has been known as one of the important indicators of the thrombosis, the formation of a blood clot inside a blood vessel or a heart. Therefore, the sensor that can detect thrombin concentration sensitively is very useful and necessary in order to save peoples’ lives when they have the thrombosis.
This thesis describes the fabrication procedure and characteristics of a new aptamer-based electrochemical immunosensor on the patterned ZnO nanorods for detecting thrombin. Immunosensors often employ a sandwich enzyme-linked immunosorbent assay (ELISA) method because of advantages such as high sensitivity, adaptability and simple detecting method. A typical sandwich ELISA immunosensor contains the capture antibody, the antigen or protein to be detected, and the detection antibody conjugated with an electrochemical or optical enzyme for signal detection. The proposed aptamer-based electrochemical immunosensor employed an aptamer labeled with Fc instead of the capture antibody of a typical sandwich ELISA method, resulting in antibody-antigen-aptamer sandwich structure. Thrombin was selected as a target protein to confirm the feasibility of the present immunosensor. An antibody-antigen-aptamer sandwich structure was fabricated by the antibody and aptamer labeled with Fc, each of which binds to two different positions of thrombin. This sandwich structure was immobilized on the ZnO nanorods electrode grown by a hydrothermal method and patterned by a lift-off technique. The characteristics of this immunosensor were evaluated by cyclic voltammetry (CV), which measures the electrochemical redox currents of the fabricated aptamer-based immunosensors at various thrombin concentrations. The reaction of the Fc-labeled aptamer improved the sensitivity and detection limit of the fabricated electrochemical aptamer-based sensors compared to other reported aptasensors. A good linearity was observed between the peak oxidation current and the logarithmic thrombin concentration in the range of 100 pM ~ 250 nM, and the detection limit of the fabricated immunosensors with ZnO nanorods electrode was about 91.04 pM thrombin. A good selectivity was also confirmed by examining the aptamer-based immunosensors in a mixture solution containing both thrombin and other proteins.